Pharmaceutical composition having prophylactic effects on lamivudine-related disease relapse and drug resistance and methods of using the same

ABSTRACT

The present invention provides a method for treating patients with liver diseases by co-administrating a pharmaceutical composition and lamivudine to the patients. The pharmaceutical composition is a herbal mixture which contains, as the core ingredients, the aqueous extracts of the entire plant of  Herba Hedyotidis diffusae,  the rhizome of Rhizoma Bistortae, the rhizome of Rhizoma Polygoni Cuspidati, and the ripe fruit of Fructus Schisandrae. The present invention also provides methods for preventing a relapse of hepatitis in patients after the lamivudine treatment has been discontinued and for suppressing the development of lamivudine drug resistance in patients by providing the lamivudine-treated patients with the pharmaceutical composition of the present invention.

RELATED INVENTION

[0001] This application claims the priority of U.S. ProvisionalApplication Serial No. 60/327,002, filed on Oct. 5, 2001, and No.60/331,257, filed on Nov. 13, 2001, which are herein incorporated byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for treatment ofpatients with liver diseases, which includes the administration topatients a drug regimen which contains a pharmaceutical composition andlamivudine. The pharmaceutical composition contains, as the coreingredients, aqueous extracts of the entire plant of Herba Hedyotidisdiffusae, the rhizome of Rhizoma Bistortae, the rhizome of RhizomaPolygoni Cuspidati, and the ripe fruit of Fructus Schisandrae. Thepresent invention also relates to methods for prevention of a relapse ofliver diseases caused by withdrawal of lamivudine treatment andsuppression of the development of lamivudine drug resistance byadminstering to patients the pharmaceutical composition of the presentinvention.

BACKGROUND OF THE INVENTION

[0003] Viral hepatitis is a mostly enterically transmitted liver diseasecaused by viral infection. The major transmission path of the disease isthrough ingestion; viral hepatitis is also transmitted through bloodtransfusion of virus-contaminated blood or blood products such as bloodplasma.

[0004] Viral hepatitis is widespread around the world. For example,there are approximately thirty million viral hepatitis patients in Chinaincluding an estimated number of nine million new patients each year andabout one hundred million hepatitis B virus (HBV) carriers. Ten percentof the pregnant women in China are estimated to be HBV carriers. Aboutone hundred thousand people in China die each year of liver canceroriginated from liver diseases.

[0005] HBV infection is caused by hepatitis B virus, which is a 42-nm,double-stranded, and circular deoxyribonucleic acid (DNA) virusbelonging to the class of Hepadnaviridae. HBV consists of a surface anda core. Four major polypeptide-encoding genes are in the HBV DNA genome:S (surface), C (core), P (polymerase), and X (transcriptionaltransactivating proteins). The S gene consists of three regions: thepre-S1 region, the pre-S2 region, and the region encoding the surfaceproteins. The surface proteins constitute the surface antigen (HBsAg).The C gene is divided into two (2) regions, the pre-core and the coreregion; the C gene encodes two (2) different proteins, the core antigen(HBcAg) and the e antigen (HBeAg).

[0006] HBsAg is found on the surface of the virus and produced in excessamount. HBsAg circulates in blood as a 22-nm spherical and tubularparticle. HBsAg can be identified in serum 30-60 days after exposure toHBV and persists for variable period of time. Antibody to HBsAg(Anti-HBs) develops after a resolved infection and is responsible forlong-term immunity.

[0007] Antibody to HBcAg (anti-HBc) develops in all types of HBVinfections and persists indefinitely. IgM anti-HBc appears early ininfection and persists for equal to or greater than six (6) months, andthus, it is a reliable marker for acute or recent HBV infection.

[0008] A third antigen, HBeAg may be detected in samples from patientswith either acute or chronic HBV infection. The presence of HBeAgcorrelates with viral replication and high infectivity. Antibody toHBeAg (anti-HBe) develops in most HBV infections and correlates with theloss of replicating virus and lower infectivity.

[0009] Hepatitis B infection progresses with varying outcomes in thepatient. Initially, acute infection in the liver causes the body tomount an immune response to get rid of the virus. The immune systemtries to clear the virus by destroying HBV infected liver cells. In somepeople, the immune response succeeds so that the virus along with theinfected liver cells are completely destroyed, and the patient makes acomplete recovery. This is called the resolution of the disease. Inother patients, however, the immune response to the infection isinsufficient to get rid of the virus entirely for many months or yearsor not at all, and the immune response slowly destroys more and moreinfected liver cells as the virus spreads, which forms long-terminfection. This slow but persistent destruction of liver cells by theimmune system leads to fibrosis, cirrhosis and even liver cancer. Peopleinfected with HBV whose immune systems cannot get rid of the virus arereferred to as the chronic HBV carriers. There are usually no obviousphysical symptoms in patients of chronic HBV. Specific blood tests willreveal the presence of the virus, and the patient is also contagious viablood, birth, sex, needles, etc. Chronic HBV carriers can pass the virusto others.

[0010] U.S. Pat. No. 5,047,407 discloses a chemical compound havingantiviral activities. The compound is (2R,cis)-4-amino-1-(2-hydroxmethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one(also known as 3TC), and given the pharmacopoeia name lamivudine andselling in the market under the trade name Epivir by Glaxo SmithKline.Lamivudine is used for treatment and prophylaxis of viral infections.

[0011] Lamivudine is a nucleoside analogue that has a potent inhibitoryeffect on RNA-dependent DNA polymerase of HBV and human immunodeficiencyvirus (HIV). The antiviral effects of lamivudine on the suppression ofHBV replication have been shown in vitro and in vivo. (Lai et al., N.Engl. J. Med. (1998), 339:61-68). Lamivudine accomplishes in mostpatients the suppression of viral replication as determined by thenegativity of serum HBV-DNA, but the compound is much less effective inclearance of HBeAg within a short period of treatment. Theineffectiveness or reduced effect on HBeAg clearance may be attributedto the persistence of HBV covalently closed circular DNA (cccDNA) inhepatocyte nuclei; rapid relapse of HBV replication often occurs afterwithdrawal of lamivudine treatment. Prolonged lamivudine treatment hasbeen proposed to solve the problem, and the seroconversion rate of HBeAghas been significantly improved.

[0012] When lamivudine is used for a prolonged period, it causesselection of lamivudine-resistant mutants of HBV and developsdrug-resistance in patients, which casts a new challenge for thetreatment regime. (Hussain et al., J. Viral. Hepat. (1999), 6:183-194;Balzarini et al., Biochem. Biophys. Res. Commun. (1996), 2:363-369).Lamivudine-resistant HBV mutants are primarily responsible for acuteexacerbation and hepatic decompensation develop in patients duringlong-term lamivudine therapy. (Liaw et al., Hepatology (1999),30:567-572).

[0013] Once the mutants are present and the patients developdrug-resistance, the physician faces a dilemma whether to stop orcontinue the treatment. In some cases, continuing treatment results inrepeated exacerbation of hepatonecroinflammation, which leads to hepaticdecompensation or cirrhosis; on the other hand, stopping treatment mayresult in re-activation of the wild type virus, which also leads toexacerbation of hepatonecroinflammation. Development of an antiviraltreatment regime against the drug-resistance HBV mutants as well as wildtype virus is pivotal for such patients.

[0014] The present invention provides an herbal pharmaceuticalcomposition that is effective for treating viral infections,particularly, the composition is effective in treating viral hepatitis.The herbal pharmaceutical composition is also effective in treatinglamivudine-resistant mutant HBV in vivo and in vitro. Additionally, theherbal pharmaceutical composition enhances the antiviral treatmenteffects of lamivudine and ameliorates the development oflamivudine-resistant HBV mutants caused by prolonged usage ofLamivudine.

[0015] The herbal pharmaceutical composition is named Yi-Gan-Kang (inChinese, “Yi” means “second or B”; “Gan” means “liver”; “Kang” means“health”; collectively, “Yi-Gan-Kang” denotes “healthy liver devoid ofHBV infection”), abbreviated as YGK.

SUMMARY OF THE INVENTION

[0016] The present invention provides a method for treating patientswith liver diseases by co-administering to said patients a drug regimenwhich contains an effective amount of a pharmaceutical composition namedYGK and lamivudine. YGK contains, as the core ingredients, aqueousextracts of the following herbs: an entire plant of Herba Hedyotidisdiffusae, a rhizome of Rhizoma Bistortae, a rhizome of Rhizoma PolygoniCuspidati, and a ripe fruit of Fructus Schisandrae. YGK further containsthe aqueous extracts of the following herbs: a rhizome of RhizomaMenispermi, a root of Radix Scutellariae, a bovine biliary powder, atuber of Radix Curcumae, a ripe fruit of Fructus Crataegi, and a root ofRadix Notoginseng. Finally, YGK further contains the aqueous extracts ofthe following herbs: aqueous extracts of a ripe fruit of Fructus Lycii,a root of Radix Ginseng Rubra, a root of Radix Scorphulariae, a root ofRadix Angelicae sinensis, and a root of Radix Astragali. Examples of theaqueous solution for extracting the pharmaceutical ingredients from theherbs include, but are not limited to, water, ethanol, or a mixturethereof. The preferred aqueous solution is water.

[0017] YGK can be adminstered to patients orally and/or intravenously.For oral adminstration, the preferred dosage amount is about 0.5 to 5 gof the YGK extract, and most favorably, 1 to 3 g of YGK, per day perperson. For intravenous injection, it is preferred that the dosageamount is about 1 to 10 g of YGK, and most favorably 3-5 g of YGK, perday per person. YGK oral dosage form and intravenous injection solutionare preferred to give to patients concurrently. The YGK extract is theaqueous extracts of the herbal ingredients after being concentrated. Adetailed description of the YGK extract is provided in Example 1 of the“Detailed Description of the Invention,” infra.

[0018] Lamivudine is preferred to be administered to patients orally.The preferred dosage amount of lamivudine is about 50-500 mg, and mostfavorably 100-200 mg per day per person.

[0019] The co-adminstration of YGK and lamivudine is particularlyeffective in treating patients with liver diseases such as hepatitis,cirrhosis, and liver cancer. The co-administration of YGK and lamivudineis especially effective in treatment of liver diseases that are causedby hepatitis B viral (HBV) infection.

[0020] The present invention also provides a pharmaceutical combinationwhich contains a first dosage unit (i.e., YGK) and a second dosage unit(i.e., lamivudine). YGK is either in an oral dosage form or in anintravenous injection solution. It is preferred that YGK oral dosagedosage form is in the form of capsules. It is preferred to take about0.1 to 2 g of YGK per adminstration and at about 2-4 times a day. Theintravenous injection solution of YGK is at the amount of about 1 to 10g per administration and preferred to be adminstered to patients once aday.

[0021] Lamivudine is preferred to be orally adminstered to patients atabout 50 to 500 mg, and most favorably 100-200 mg per administration andat about once a day.

[0022] The present invention further provides a method for preventing arelapse of hepatitis in patients after the lamivudine treatment has beendiscontinued by administering YGK orally and/or intravenously topatients after their lamivudine treatment has been discontinued. Thismethod is particularly effective when the hepatitis is caused byhepatitis B viral (HBV) infection.

[0023] Finally, the present invention provides a method for supressingthe development of lamivudine drug resistance in patients byadminstering YGK orally and/or intravenously to patients that are takinglamivudine. This method is particularly effective when the lamivudinedrug resistance is caused by development of lamivudine-resistanthepatitis B virus (HBV) strains in patients.

BRIEF DESCRIPTION OF DRAWINGS

[0024]FIG. 1 shows the effects of YGK on the production rate of HBsAg inHep3B cells (FIG. 1A) and HepA2 cells (FIG. 1B). ♦--♦ represents thecells treated with YGK at different concentrations. ▪--▪ represents thecells treated with YGK and a “conditional medium”. The conditionalmedium is the medium collected from the culture medium of monocyteswhich have been incubated for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The lamivudine-resistant viruses have a characteristic amino acidsubstitution over tyrosine-methionine-aspartate-aspartate (YMDD) motifof the RNA-dependent DNA polymerase, as was seen in lamivudine-resistantimmunodeficiency virus. (Balzarini et al., (1996), 2:363-369). Themethionine at codon 552 was either replaced by an isoleucine (M552I) ora valine (M552V). (Ling et al., Hepatology (1996), 3:711-713; Tipples etal., Hepatology, 3:714-717; and Naoumov et al., Hepatology (1996),24:282; which are herein incorporated by reference).

[0026] The M552V mutation was frequently accompanied by aleucine528-to-methionine (L528M) substitution. (Fu et al., Biochem.Pharmacol. (1998), 55:1567-1572; Niesters et al., J. Infect. Dis.(1998), 177:1382-1385). These two mutants, M552I and L528M/M552V, werecommonly identified lamivudine-resistant mutants. (Liaw et al.,Hepatology (1999), 30:567-572). In addition, two additional resistantmutants, L528M/M552V and A529T were developed. Strikingly, replicationof some of these mutants was found to be partially lamivudine-dependent.

[0027] The pharmaceutical composition of the present invention, YGK,contains fifteen (15) herbs, which are grouped according to theiressential roles as follows:

[0028] First, there are four (4) herbs which form the core ingredientsof YGK. These 4 herbs contributes to the primary efficacy and healingeffects of the composition. The 4 herbs are: (1) diffusehedyotis/spreading hedyotis (Pharmaceutical name: Herba Hedyotidisdiffusae; Botanical name: Hedyotis diffusa Willd.); (2) bistort rhizome(Pharmaceutical name: Rhizoma Bistortae; Botanical name: Polygonumbistorta L.); (3) giant knotweed rhizome (Pharmaceutical name: RhizomaPolygoni Cuspidati; Botanical name: Polygonum cuspidatum Sieb. etZucc.); and (4) Chinese magnoliavine fruit (Pharmaceutical name: FructusSchisandrae Chinensis; Botanical name: Schisandra chinensis (Turcz.)Baill., S. sphenanthera Rehd. et Wils.). The preferred weight ratio ofdiffuse hedyotis, bistort rhizome, giant knotweed rhizome, and Chinesemagnoliavine fruit is about 3:3:1:2.

[0029] The function of the core herbs function includes clearing heatand toxic substances while improving immune system and circulation,curing symptoms of jaundice, and having beneficial effect on internalorgans.

[0030] There are six (6) additional herbs that are used to improve andbalance the pharmaceutical activities produced by the core ingredients.These six herbs also have toning effect and improve blood circulation inthe liver to help the curing of the liver diseases. The six ingredientsare: (1) Asiatic moonseed rhizome (Pharmaceutical name: RhizomaMenispermi; Botanical name: Menisermum dauricum DC); (2) baical skullcaproot (Pharmaceutical name: Radix Scutellariae; Botanical name:Scutellaria baicalensis Georgi); (3) bovine biliary powder (Zoologicalname: Vesica Fellea Bovus); (4) tumeric root-tuber (Pharmaceutical name:Radix Curcumae; Botanical name: Curcuma wenyujin Y. H. Lee et C l Ling);(5) Hawthorn Fruit (Pharmaceutical name: Fructus Crataegi; Botanicalname: Crataegus pinnatifida Bge.); and (6) sanqui (Pharmaceutical name:Radix Notoginseng; Botanical name: Panax notoginseng (Burk.)).

[0031] Finally, there are additional five (5) ingredients which are usedto primarily provide nutrients and energy sources for patients toexpedite the recovery process. These ingredients include: (1) barbarywolfberry fruit (Pharmaceutical name: Fructus Lycii; Botanical name:Lycium barbarum L.); (2) red ginseng (Pharmaceutical name: Radix GinsengRubra; Botanical name: Panax Ginseng C. A. Mey); (3) figwort root(Pharmaceutical name: Radix Scrophulariae; Botanical name: Scrophularianingpoensis); (4) Chinese angelica (Pharmaceutical name: Radix Angelicaesinensis; Botanical name: Angelica sinensis (Oliv.) Diels); and (5)milkvetch root (Pharmaceutical name: Radix Astragali; Botanical name:Astragalus membranaceus (Fisch.) Bge.).

[0032] Among the five ingredients, red ginseng and milkvetch root alsohave the capacity of improving immune functions of the body to fense offdiseases.

[0033] The pharmaceutical names, botanical or zoological names, familynames, common descriptions, and major ingredients of the herbs used inthe present invention is shown in Table 1. TABLE 1 Herbs of the PresentPharmaceutical Composition Pharmaceutical Botanical/Zoo- Common MajorName logical Name Family Description Ingredients Herba HedyotidisHedyotis diffusa Rubiaceae hedyotis, hentriacontane, Diffusae (Willd.)Roxb., oldenlandia stigmastatrienol, also known as ursolic acid,Oldenlandia oleanolic acid, β- difusa sitosterol, ρ- coumaric, β-sitosterol-D- glucoside Radix et Rhizoma Polygonum Polygonaceae GiantKnotweed emodin, chryso- Polygoni cuspidatum root and phanol, rheicCuspidati Sieb. et Zucc. Rhizome acid, emodin monomethyl ether,polygonim, and physcion-8-β-D- glucoside Rhizoma Polygonum PolygonaceaeBistort Rhizome n/a Bistortae bistorta L. Rhizoma MenispermumMenispermaceae Asiatic n/a Menispermi dauricum DC. Moonseed RhizomeRadix Scutellaria Labiatae Baical Skullcap baicalein, Scutellariaebaicalensis Root baicalin, Baicalensis Georgi wogonin, wogonoside,neobaicalein, oroxylin aglucuronide, camphesterol, β- sitosterol,benzoic acid Vesica Fellea Bovine Biliary n/a Bovus powder RadixAstragali Astragalus Leguminosae Milkvetch Root D-β-asparagine,membranaceus 2′,4′-dihydroxy- (Fisch.) Bge. 5,6- var. dimethoxyisoflavmongholicus. ane, calycosin, (Bge.) Hsiao or formononetin, Astragaluscycloastragenol, membranaceus astragalosides, (Fisch.) Bge. choline,betaine, kumatakenin, sucrose, glucoronic acid, β-sitosterol FructusLycii Lycium Solanaceae Barbary betaine, carotene, barbarum L. WolfberryFruit physalien, thiamine, riboflavin, vitamin C, β- sitosterol,linoleic acid Radix Panax noto- Araliaceae San-chi, Arasaponin A,Notoginseng ginseng (Burk.) notoginseng, arasaponin B, F.H. chen, P.Tian qi, Shen san dencichine pseudoginseng qi Wall, P. sanchi Hoo. RadixGinseng Panax Ginseng Araliaceae Red Ginseng Panaxatriol, Rubra C.A. MeyPanaxadiol, Other Panoxisides, Panoquilon, Panaxin, Ginsenin, α-Panaxin, Protopanaxadiol, Protopanaxtriol, Panacene, Panaxynol, PanaenicAcid, Panose, Dammarane, Glucose, Fructose, Maltose, Sucrose, NicrotinicAcid, Riboflavin, Thiamine Radix Scrophularia Scrophulariaceae FigwortRoot, 1-asparagine, Scrophulariae ningpoensis Scrophularia oleic acid,Ningpoensis Hemsl. or S. linoleic acid, buergeriana stearic acid, Miq.carotene Fructus Schisandra Magnoliaceae Chinese sesquicarene, β-Schisandrae chinensis Magnoliavine bisabolene, β- Chinensis (Turcz.)Baill., Fruit, schisandra chamigrene, α- S. sphenanthera fruit ylangene,Rehd. et Wils. schizandrin, pseudo-γ- schizandrin, deoxyschizandrin,schizandrol, citral, stigmasterol, vitamin C, vitamin E Tuber CurcumaeCurcuma Zingiberaceae Turmeric Root- d-camphene, d- wenyujin Y. H.tuber, curcuma camphor, 1-α- Lee et C. Ling., curcumene, 1-β- or Curcumacurcumene, Longa L., or curcumin, Curcuma demethoxycurcu aromatica min,Salisb., or bisdemethoxycur Curcuma cumin, zedoaria Rosc., turmerone,ar- or Curcuma turmerone, kwangsiensis S. carvone, ρ- C. Lee et C. F.tolylmethylcarbi Liang noldiferuloylmet hane Fructus Crataegi CrataegusRosaceae Hawthorn Fruit crategolic acid, pinnatifida Bge., citric acid,C. pinnatifida tartaric acid, Bge. var. major flavone, sugars, N.E. Br.or C. glycosides, suneata Sieb. et vitamin C Zucc. Radix AngelicaeAngelica Umbellferae Chinese butylidene Sinensis sinensis (Oliv.)Angelica root, phthalide, Diels tang-kuei ligustilide, n- butylidene-phthalide, sequiterpenes, carvacrol, dihydroplithalic anhydride,sucrose, vitamin B₁₂, carotene, β- sitosterol

[0034] Diffuse hedyotis or spreading hedyotis (Herba HedyotidisDiffusae) belongs to the family of Rubiaceae. The entire plant is usedas an herbal medicinal component. The herb has no toxicity. The herb isharvested in summer and autumn in mainland China and in late spring orearly winter in Taiwan. In “Materia Medica” (Chinese Herbal medicine),compiled and translated by Dan Bensky & Andrew Gamble, diffusehedyotidis clears heat and resolves dampness by promoting urination. Itis particularly useful for relieving hot painful urinary dysfunction anddamp-heat jaundice. Diffuse hedyotidis is the major ingredient in thepresent herbal pharmaceutical composition which contributes to themedicinal effect on liver diseases and HIV.

[0035] Bistort rhizome (Rhizoma Bistortae) is the dried rhizome of theplant Polygonum bistorta L. It belongs to the family of Polygonaceae.Bistort rhizome has moderate cool property (meaning that bistor rhizomeis an “yang” herb). It can be used to remove toxic heat, to promote thesubsidence of swelling and to stop bleeding.

[0036] Giant knotweed rhizome (Radix et Rhizoma Polygoni Cuspidati) isthe dried rhizome and root of polygonum cuspidatum Sieb. et Zucc. Itbelongs to the family of Polygonaceae. The plant is grown throughoutChina, especially Jiangsu, Zhejiang, Anhui, Guangdong, Guangxi, Sichuan,and Guizhou provinces. The plant is harvested in spring and autumn.Giant knotweed rhizome is normally used to dispel damp, to eliminateblood stasis and alleviate pain, to relieve cough, and to resolvephlegm.

[0037] Chinese magnoliavine fruit (Fructus Schisandrae) is the driedripe fruit of Schisandra chinensis (Turcz.) Baill. or Schisandrasphenanthera Rehd. et Wils. It belongs to the family of Magnoliaceae.The former, the best of its kind, is produced in northern parts of Chinaand is habitually called “Northern schisandra fruit”; the latter iscommonly referred to as the “Southern schisandra fruit” as it isproduced in the southern parts of China. Both kinds can be used for thepharmaceutical preparation of the present invention. The fruit iscollected in autumn and dried under the sun after removing the fruitstalks. Chinese magnoliavine fruit is generally used to arrestdischarges, replenish qi, promote fluid secretion, tonify the kidney,and induce sedation. Chinese magnoliavine fruit can also decrease thelevel of GPT (glutamate-pyruvate transaminase) in patients withhepatitis.

[0038] Asiatic moonseed rhizome (Rhizoma Menispermi) is the driedrhizome of Menispermum dauricum DC. It belongs to the family ofMenispermaceae. Asiatic moonseed rhizome has cool property. It can beused to remove toxic heat and relieve rheumatic pains.

[0039] Baical skullcap root (Radix Scutellariae) is the dried root ofScutellaria baicalensis georgi. It belongs to the family of Labiatae.The plant is produced in the provinces of Hebei, Shanxi, Inner Mongolia,etc., and collected in spring or autumn. Baical skullcap root is used toremove damp-heat, counteract toxicity, arrest bleeding, and preventabortion, in patients.

[0040] Bovine biliary powder is the gall bladder of the cow, VesicaFellea Bovus. It can clear heat and alleviate spasms.

[0041] Turmeric root-tuber (Radix Curcumae) is the dried root tuber ofCurcuma wenyujin Y. H. Lee et C. Ling., or Curcuma Longa L., or Curcumaaromatica Salisb., or Curcuma zedoaria Rosc., or Curcuma kwangsiensis S.G. Lee et C. F. Liang. The herb is mainly produced in Sichuan, Zhejiang,Guangdong, and Guangxi provinces in China, and harvested in winter orspring, washed clean after the removal of the hairy rootlets, boiledthoroughly, and dried in the sun. It belongs to the family ofZingiberaceae. Turmeric root-tuber tastes bitter and had cool property.It can be used to clear heat, alleviate spasms and chest pain, andresolve phlegm.

[0042] Hawthorn fruit (Fructus Crataegi) is the dried ripe fruit ofCrataegus pinnatifida Bge. var major N. E. Br., or Crataegus pinnatifidaBge., or Crataegus cuneata Sieb. It is produced primarily in Henan,Jiangsu, and Shandong provinces of China. It is harvested in autumn,sliced, and dried in sunlight. It belongs to the family of Rosaceae.Hawthorn fruit is normally used to stimulate digestion and promote thefunctional activity of the stomach. It can also improve the normal bloodflow and dissipate blood stasis.

[0043] Sanqi, or San-chi, (Radix Notoginseng) belong to the family ofAraliaceae. Sanchi (Sanqi) is the dried root of Panax notoginseng(Burk.) F. H. Chen. The plant is also known as P. pseudoginseng Wall andP. sanchi Hoo. The plant grows in Yunnan, Guangxi, Sichuan, Guizhou, andJiangxi provinces of China, and is harvested in the autumn or winter ofthe third or seventh year, either before the flowers bloom (better) orafter the fruit is ripe. H. Gao et al., Pharmaceutical Research, (1996)13(8): 1196-1200, disclose that polysaccharides from Panax notoginseng(San-Chi) have immuno-stimulating activities in vitro.

[0044] Barbary wolfberry fruit (Fructus Lycii) is the dried ripe fruitof Lycium barbarum L. It belongs to the family of Solanaceae. The plantis mainly produced in Ningxia, Gansu, and Qinghai provinces of China. Itis harvested in summer and autumn. It nourishes and tonifies the liverand kidneys. It can also replenish vital essence and improve eyesight.

[0045] Figwort Root (Radix Scrophulariae) is the dried root ofScrophularia ningpoensis Hemsl. It belongs to the family ofScrophulariaceae. The herb is chiefly produced in Zhejiang and Sichuanprovinces of China and harvested in winter when the part of the plantabove-ground has withered. The roots are piled and dried in sunlightalternately until the inside becomes black and then sliced for use.Figwort root can reduce heat from blood. It also has nourishing capacityand can counteract toxicity.

[0046] Red ginseng (Radix Ginseng Rubra) is the steamed and dried rootof the cultivated form of Panax ginseng C. A. Mey (commonly known as“Yuanshen”). The herb turns red after being steamed and its propertiesbecome warmer in nature. It belongs to the family of Araliaceae. Thepharmaceutical effects of ginseng are in its dried root. Ginseng haseffects on central nervous system. It enhances both stimulatory andinhibitory processes in the central nervous system, thereby improvingthe adaptability of nervous responses. Ginseng can also lower serumglucose and cholesterol. It also shows therapeutic and preventive effecton peptic ulcer.

[0047] Chinese angelica (Radix Angelicae Sinensis) is the dried root ofAngelica sinensis (Oliv.) Diels. It belongs to the family ofUmbelliferae. The herb is mainly produced in Gansu and Shanxi provincesof China. It is harvested in late autumn, smoked dry on slow fire aftergetting rid of the rootlets, sliced, or stir-baked with wine. Chineseangelica can enrich blood, promote blood circulation, regulatemenstruation, relieve pain, and relax bowels.

[0048] Milkvetch root (Radix Astragali) is the dried root of Astragalusmembranaceus (Fisch.) Bge. var. mongholicus. (Bge.) Hsiao or Astragalusmembranaceus (Fisch.) Bge. It belongs to the family of Leguminosae. Theherb is mainly produced in Shanxi, Gansu, Heilongjiang, and InnerMongolia of China. The plant of four-year old or older is harvested inspring or autumn. Milkvetch root can promote discharge of pus and thegrowth of new tissue.

[0049] YGK is particularly effective in treating patients with liverdiseases. YGK is suitable for preparation in a scale typical forpharmaceutical industry as well as for smaller measure.

[0050] In the process for making the herbal composition of the presentinvention, the individual herbal components are pretreated according tothe common procedures. The herbs are cut and put in a container withwater and/or lower alcohol (such as ethanol) to boil and simmer twice.The first time of simmering takes about two hours, the solution iscollected, and water and/or lower alcohol is added for the second timeof simmering for about 1.5 hour. The solutions from the simmering stepsare collected by passing through a sieve/filter. The filtrate is thencondensed from about 1.4 fold by volume to 1.0 fold by volume.Subsequently, the liquid condensate is spray-dried and granulated toform particles. The particles are further packaged into capsules andpreserved for use or for further analysis by the conventional means ofthe active ingredients to ensure their quality. The liquid condensatecan also be made for intravenous injection solution. Approximately 5 mlof intravenous injection solution is in an ampoule.

[0051] The composition of the present invention can further be processedand formulated in a form suitable for oral administration or intravenousinjection.

[0052] In the clinical research on the effects of YGK in treatingchronic hepatitis B, the curing effects were valued at:

[0053] (1) Continued disappearance of serum HBV-DNA, which is anindication of the clearance of HBV-DNA in serum and liver tissue;

[0054] (2) Clearance of HBsAg and production of anti-HBs;

[0055] (3) Suppression of HBV replication;

[0056] (4) Clearance of HBeAg and production of anti-HBe; and

[0057] (5) Significant improvement of liver function (as judging bytissue damage) as shown by the normal level of serum aminotransferase,histological examination of dead liver cells, and indications ofalleviated inflammation.

[0058] When co-administered with lamivudine, YGK is especially effectivein curing chronic hepatitis B with quick clearance of serum HBV-DNA andnegativity of HBeAg and HBsAg. To be effective for the treatment, YGK isadministered into patients in two forms, capsule or intravenousinjection solution. It can be administered separately or together withlamivudine. The herbal composition of the present invention has alsodemonstrated capability of normalizing hepatic enzymes and alleviatingclinical symptoms in patients with hepatitis or cirrhosis.

[0059] The methods of the present invention are especially effective incuring chronic hepatitis B with quick clearance of serum HBV-DNA andnegativity of HBeAg and HBsAg. The method of the present invention fortreating hepatitis B includes concurrent- or co-administration of YGKwith lamivudine is effective to enhance the anti-viral properties oflamivudine and to suppress the development of lamivudine-resistant HBVmutants caused by prolonged usage of lamivudine.

[0060] The combined treatment of YGK and lamivudine of the presentinvention can achieve the dual goals of suppressing viral replicationand improving immune system function of the patient.

[0061] Lamivudine, a nucleoside analogue, is effective on the inhibitionof enzymatic activity of reverse transcriptase during HBV replication.Six months of lamivudine treatment is effective to decrease HBV-DNA inserum, and the rate of patients turning negative can reach as high as90.4%. The shortcomings associated with lamivudine treatment are withthe cut-off of a single phase in viral replication cycle by lamivudine,the rate for patients turning negative for HBeAg is very low, and a longperiod of lamivudine treatment is then needed. After the completion ofthe lamivudine treatment, the patients have a high rate of turningpositive for HBeAg.

[0062] The two types of YGK formulations, i.e., YGK intravenousinjection solution and YGK capsules, have slightly different anti-HBVeffects. YGK intravenous injection solution has direct anti-HBV effect.It can block the takeup of nucleotides by the virus during viralreplication so that the virus will become feeble and die. YGK capsules,on the other hand, are particularly effective for protecting the livercells and adjusting the immune function. The combined use of theintravenous injection solution and capsules of YGK is effective forsuppression of HBV and recovery of liver function.

[0063] The present invention demonstrates that the combined treatment oflamivudine and YGK has multiple functions on multiple phases of HBVreplication: Lamivudine inhibits the enzymatic activity of reversetranscriptase during HBV replication, and YGK intravenous injectionsolution blocks the take-up of nucleotides by the virus during DNAreplication; at the same time, YGK capsule adjusts the patient's immunefunction. The effective combination can quickly clear the serum HBV-DNAwithin 8 weeks so that 100% of the patients show negative for HBV-DNA,and more patients turn negative for HBeAg after 16 weeks of treatment.Lamivudine treatment in the combined treatment is stopped after 16 weeksof treatment so that the period of taking lamivudine is greatly reducedand the side effects of prolonged lamivudine treatment are avoidedincluding induction of lamivudine-resistant HBV and relapse of thedisease.

[0064] The following example is illustrative, but not limiting the scopeof the present invention. Reasonable variations, such as those occur toreasonable artisan, can be made herein without departing from the scopeof the present invention.

EXAMPLE 1 Pharmaceutical Preparation

[0065] The kinds and amounts of the herbal ingredients used in theprocess of making the pharmaceutical composition of the presentinvention are described in Table 2. The pharmaceutical composition iscalled “YGK.” It can be formulated as injection solution and capsules.TABLE 2 Ingredients Used In Making YGK Injection Solution and YGKCapsules Component Amount (kg) Diffuse heydyotis 2.49 Bistort Rhizome2.49 Giant Knotweed root and 0.83 Rhizome Asiatic Moonseed Rhizome 0.83Baical Skullcap Root 0.83 Bovine Biliary powder 0.083 Milkvetch Root1.66 Barbary Wolfberry Fruit 2.49 Sanchi 0.83 Red Ginseng 0.83 Figwortroot 1.66 Chinese Magnoliavine Fruit 1.66 Turmeric Root-tuber 0.83Hawthorn fruit 1.66 Chinese Angelica 0.83

[0066] (1) Quality Controls of Raw Materials

[0067] Quality control tests carried out for each individual rawmaterials according to conventional methods used in the herbalpharmaceutical field which include, but are not limited to, physicalappearance, loss on drying, total ash, acid insoluble ash, alcoholextracts, water extracts, TLC, HPLC, heavy metals, microbial counts andresidual pesticides. Bovine biliary powder was tested for appearance,TLC and general chemistry.

[0068] (2) Manufacturing Process

[0069] The individual herbal components were pretreated according tocommon procedures. The herbs were weighed according to Table 3. Aflowchart of the manufacturing process for making YGK capsules and/orinjection solution is provided in Table 3: TABLE 3 YGK ManufacturingProcess Manufacturing Process Quality Control Procedure Raw HerbsDelivered Quality Control of Raw Herbs:

•Physical Appearance Preparation (cutting, drying, etc.)

Quality Control of Raw Herbs: •Physical Appearance •Loss on DryingObtain Total of 20.003 kg Raw Herbs •Total Ash and Put into Extractorfor •Acid Insoluble Ash Manufacturing •Alcohol Extracts

•Water Extracts •TLC •HPLC Add 350 L ± 10% Water to Soak •Heavy MetalsHerbs in Extractor for 60 ± 10 mm •Microbial

Residues Pesticide Note: Bovine biliary powder (also known as BovisBezoar) is only tested for appearance, TLC and General Chemistry 1^(st)Extraction Parameter Set: 1. Dial Set Temperature at 95° C. In-ProcessQuality Control: with the Acceptable Range of (take 10˜15 g as testsample) 90° C.˜100° C. • Concentration of Solid 2. Dial Set SteamPressure at Content 2kg/cm2. • Concentration of Water 3. Dial Read Outthe Lid Pressure at Content 0.2˜O.4 kgf/cm² Range. 4. Extract for 45minutes.

1^(st) Concentration Parameter Set: 1. Dial Read Out Vacuum at −60˜ −76cmHg. 2. Dial Read Out Temperature at 40° C. ± 5° C. 3. Process for 40minutes.

2^(nd) Extraction Parameter Set: 1. Dial Set Temperature at 95° C. withthe Acceptable Range of 90° C.˜100° C. 2. Dial Set Steam Pressure at2kg/cm². 3. Dial Read Out the Lid Pressure at 0.2˜0.4 kgf/cm^(f) Range.4. Extract for 45 minutes

2^(nd) Concentration Parameter Set: 1. Dial Read Out Vacuum at −60˜ −76cmHg. 2. Dial Read Out Temperature at 40° C. ±5° C. 3. Process for 40minutes.

Combined Concentrated Extracts and Pour into the Stainless Container

Spraying Silo Parameter Set: 1. Dial Set Temperature at 60° C. 2.Pre-heat for 15 minutes.

Fluid Bed Dryer 1. Add 3.9 kg of Starch (adj. Base on water content ofthe starch). 2. Dial Set In-Temperature at 60° C; Out-Temperature at 50°C. 3. Pre-heat for 10 minutes. 4. Process for target of 2 hours ± 20min. until LOD<5%. (continued on next page)

Sievinig Parameter Set: 1. Dial Read out Temperature at 23 ± 4° C. 2.Dial Read out RH at 50 ± 10%. 3. Sieve with #100 followed by #80.

Quality Control of Drug YGK Powder Substance: •Physical Appearance •Losson Drying •Total Ash •Acid Insoluble Ash •Alcohol Extracts •WaterExtracts •TLC •HPLC •Heavy Metals •Microbial •Residues Pesticide•Stability

[0070] The individual herb was cut into small pieces and thoroughlymixed together. The mixed herbs were placed in bags with sufficientspace to spread out. The bags were placed in an extractor with 350 L(±10%) of water and soaked for about 60±10 min. The herbs were firstextracted at 95±5° C. under steam pressure of 2 kg/cm² and lid pressureof 0.2-0.4 kg/cm² for 45 min. The water extract was collected into aconcentrator and concentrated at 40±5° C. under vacuum of −60 to −76cmHg for 40 min to form the first concentrate.

[0071] The herbs in the bags were recovered from the extractor, placedinto another 350 L (±10%) of water and extracted again at 95±5° C. understeam pressure of 2 kg/cm2 and lid pressure of 0.2-0.4 kgf/cm² for 45min. The extract from the second extraction was collected into aconcentrator and concentrated at 40±5° C. under vacuum of −60 to −76cmhg for 40 min to form the second concentrate.

[0072] The first and second concentrates were combined and poured into astainless container. The total weight of the combined concentrates wasabout 6.1 kg±10%. The combined concentrates were called the “YGKextract.”

[0073] For the YGK injection solution, about 0.4 g of the “YGK extract”was dissolved in about 1 ml of the injection buffer. About 5 ml of theinjection solution was poured into an ampoule.

[0074] For the YGK powders (which were packaged into YGK capsule), about3.9 kg of starch (adjustable based on the water content of the starch)were added to the YGK extract and spray dried in a fluidized bed settingat in-temperature of 60° C. and out-temperature of 50° C. forapproximately 120±20 min until LOD (limit of detection) <5%.

[0075] The resultant powders were passed through a 100-mesh sieve andthen a 80-mesh sieve. The final yield of the YGK powders were about9.5˜10.5 kg. The YGK powders were further packaged into capsules. Therewere two dosage forms of YGK capsules: A 500 mg of the YGK capsule,which contained about 305 mg of the “YGK extract” and about 195 mg ofstarch; and a 220 mg of the YGK capsule, which contained about 134 mg ofthe “YGK extract” and about 86 mg of starch.

[0076] (3) In-process Quality Controls

[0077] After the extracts were concentrated, a 10-15 g sample wascollected and the concentrations of the solid content and the watercontent were determined by methods described in US Pharmacopoeia, ChinaPharmacopoeia, and/or Japanese Pharmacopoeia.

[0078] (4) Quality Control of YGK Powders

[0079] Quality control tests carried out for the YGK powders include,but are not limited to, physical appearance, loss on drying, total ash,acid insoluble ash, alcohol extracts, water extracts, TLC, HPLC, heavymetals, microbial counts, residual pesticides and stability byconventional methods and by methods described in US Pharmacopoeia, ChinaPharmacopoeia, and/or Japanese Pharmacopoeia.

[0080] (5) Quality Control of YGK Capsules

[0081] Quality control tests carried out for the 500 mg YGK capsulesinclude, but are not limited to, physical appearance, loss on drying,total ash, acid insoluble ash, alcohol extracts, water extracts, TLC,HPLC, heavy metals, microbial counts, residual pesticides and stabilityby conventional methods and by methods described in US Pharmacopoeia,China Pharmacopoeia, and/or Japanese Pharmacopoeia.

EXAMPLE 2 Efficacy of the YGK Herbal Composition on Treatment ofPatients with Hepatitis B (HBV)

[0082] The clinical research was conducted in the Liberty MilitaryHospital 211 in China. The course of hepatitis B is determined by manyfactors, including immune response, host genetic factors, and HBVmutations. The chronic hepatitis distinguishes from the acute hepatitis.The acute hepatitis is the active and symptomatic infection of theliver. A patient with the acute hepatitis is contagious. Symptoms ofacute HBV infection are non-specific, but may include malaise, anorexiaor jaundice. A chronic hepatitis patient is asymptomatic. The HBV ispresent in the liver and blood, although there are usually no obviousphysical symptoms. Specific blood tests will reveal the presence of thevirus, and the patient is also contagious via blood, birth, sex,needles, etc. Cirrhosis is the pathological dysfunctional state of theliver, the hardening of the liver as the result of chronic hepatitis,chronic persistent hepatitis (CPH) and chronic active hepatitis (CAH).

[0083] A total of 948 patients with acute HBV, chronic HBV, and livercirrhosis participated in a clinical comparative study. The patientswere divided into two (2) groups. The study group had 642 patients andthe comparative group has 306 patients. The data on patients whoparticipated in this study are listed in Table 4. TABLE 4 Patients Datain the Clinical Study Group Study Group Comparative Group Total Numberof Patients 642 306 Sex Distribution of the Male: 482 Male: 229 PatientsFemale: 160 Female: 77 Age Distribution of 7 to 74 years old 8 to 70years old Patients (average age: 32.5) (average age: 30.5) *Symptoms ofPatients' Acute Hepatitis B: 282 Acute Hepatitis B: 109 Liver DiseaseChronic Hepatitis: 276 Chronic Hepatitis Cirrhosis: 84 B: 114 Cirrhosis:83

[0084] The patients were treated according to the following regime:

[0085] (1) The patients in the study group were each orally administeredeight (8) YGK herbal composition containing the herbal composition ofthe present invention per day.

[0086] (2) The patients in the comparative group were each orallyadministered four (4) Hugang (“liver protecting”) tablets per day.Hugang (liver-protecting) tablet is made from schisandrafruit (FructusSchisandrae Chinensis) alcohol extractant, liver-protecting extractant(including Junchen, Zihu, and woad root (isatis root, Radix Isatidis seuBaphicacanthi)), and biliary powder, etc. It has an effective rate of95.08% for treating chronic hepatitis (70% with significant effect), and82.5% for treating cirrhosis (63% with significant effect).

[0087] The treatment lasts for ninety (90) days.

[0088] Table 5 shows the results of this clinical study. TABLE 5 Effectsof YGK Capsule Treatment Group Number of Patients with Positive Effect*(%) Study (642 patients) 456 (71.03%) Comparative (306 patients) 104(33.98%)

[0089] As indicated in Table 5, approximately 71.03% of patients whotook the YGK herbal composition for 90 days show positive responses tothe herbal composition. This is contrary to the comparative group wherethe patients were given a popular “liver protecting” tablets which wereavailable in the Chinese market. Patients who had taken the “liverprotecting” tablets only have an effective rate of approximately 33.98%to show improvement in their liver diseases.

EXAMPLE 3 Toxicity Study of the YGK Herbal Composition in AnimalsPurpose

[0090] The following experiment was conducted at the ToxicologyLaboratory of the Institute of Labor, Health, and Occupational Diseaseof Heilongjiang Province in China to examine acute toxicity of the YGKherbal composition during intravenous injection in animals.

[0091] Methods:

[0092] Experimental animals were Japanese big-ear white rabbits obtainedfrom the Animal Center of Haerbin Medical University in Haerbin,Heilongjiang Province, China. These rabbits were characterized by theobvious blood vessels on ears which facilitates the operation ofinjection during the experiments.

[0093] Ten (10) rabbits were obtained including six (6) males and four(4) females, each weighing between 1900 g to 3000 g.

[0094] The rabbits were randomly divided into two (2) groups, fiverabbits in each group including two (2) females and three (3) males. TheYGK herbal composition was intravenously injected into the rabbitsthrough the veins on their ears at dosages of 10 g/kg and 15 g/kg,respectively, for two groups.

[0095] The concentration of injection fluid containing the herbalcomposition was about 1 g/ml. So the higher dosage group at 15 g/kg hasa concentration of about 15 ml/kg, which could be calibrated as a sixty(60) kg-weighted adult who was treated by 900 ml of the herbalcomposition at a time.

[0096] The rabbits were observed for behaviour continuously for a periodof two (2) weeks after intravenous injections. Observation was conductedhourly at day 1; during the following days, observation was conductedfour-six (4-6) times per day.

[0097] At the end of the observation period, rabbits were sacrificed anddissected to examine the eyes, liver, lung, and spleen for adverseeffects.

[0098] Results:

[0099] No abnormal behavior was observed of the rabbits during theobservation period. The rabbits showed normal body weight increaseduring the period. After the sacrifice and dissection, inspection of theeyes, liver, lung, and spleen showed no extraordinary syndromes. Theresults when compared to a general acute toxicity index were normal andno acute toxicity.

EXAMPLE 4 In Vitro Anti-viral Activity of YGK

[0100] The production of HBsAg by human hepatoma Hep3B cells was verysensitive to various agents. (Hsu et al., J. Biol. Chem. (1993),268:23093-23097). Thus, Hep3B cells could be developed as an effectiveassay system for examining antiviral activity associated Chinese herbmedicine.

[0101] In order to further study the regulation of gene expression andviral replication of HBV, another hepatoma cell line, HepA2, wasdeveloped. HepA2 cell was derived from HepG2 cell by transfection oftandem arranged HBV genome into HepG2 cell which contains no HBVsequence in its genome. HepA2 cell could synthesize and secrete bothHBsAg and HBeAg into culture medium.

[0102] These two cell lines were used as a model system to examine theantiviral activity of YGK. Because YGK has been used to directly treatpatient with chronic active hepatitis by i.v. injection, the possibilitythat YGK could cooperate with cytokines secreted by the monocytes incirculation to inhibit HBsAg production by liver cells was also testedby collecting the culture medium from monocytes (from blood) incubatedfor 24 h. The monocyte-incubated cultured medium was named as the“conditional medium.”

[0103] The Hep3B cells and HepA2 cells were then treated with either YGKalone or YGK with the conditional medium. The results are shown in FIG.1.

[0104] The inhibitory effect of YKG alone on HBsAg production in bothHep3B and HepA2 cells was dose dependent. (♦--♦ in FIG. 1A). About 50%of HBsAg production was inhibited in Hep3B cells when YKG was at aconcentration of about 2% (w/v). About 50% of HBsAg production wereinhibited when YGK was at a concentration of about 3%.

[0105] In Hep3B cells, the conditional medium alone has no effect onHBsAg production. (Not shown in FIG. 1A). However, when the conditionalmedium was added to the culture medium for Hep3B cells, together with 2%of YGK, the production rate of HBsAg in Hep3B cells decreased from about50% (without the conditional medium) (♦--♦) to about 15% (with theconditional medium). (▪--▪ of FIG. 1A). The inhibitory effect of HBsAgproduction appeared to be plateaued at about 4% YGK, where the HBsAgproduction rate was at about 5%.

[0106] In HepA2 cells, the conditional medium alone appeared to havesome effect on HBsAg production. (Not shown in FIG. 1B). When theconditional medium was added to the culture medium for Hep3B cells,together with 2% of YGK, the production rate of HBsAg in Hep3B cellsdecreased from about 65% (without the conditional medium) to about 20%(with the conditional medium). (▪--▪ of FIG. 1A). The inhibitory effectof HBsAg production appeared to be plateaued at about 3-5% YGK, wherethe HBsAg production rate was at about 10%.

EXAMPLE 5 In Vitro Anti-Viral Studies of The Effects of YKG onInhibiting The Viral Replication of lamivudine-resistant HBV Mutants

[0107] Methods:

[0108] (1) Site-Directed Mutagenesis

[0109] The methods to generate plasmids containing HBV genomes areaccording to Yeh et al. (Yeh et al., Hepatology (2000), 31:1318-1326).Briefly, a plasmid, pCMVHBV, was generated by inserting one copy ofgreater-than-unit-length HBV genome (3.37kb; nucleotides 1820 to1990;adw subtype, derived from pECE-C [Walsh et al., Gut (2001),49:436-440]) into a vector, pRc/CMV (Invitrogen, SanDiego, Calif.). AHind III site and an Xba I site were engineered at the 3′ and 5′ ends ofthis insert.

[0110] To generate plasmids expressing lamivudine resistance mutants,site-directed mutagenesis experiments were carried out using a PCR-basedstrategy. Two primers, Pol-L(upstream) and Pol-R (downstream), whichflanked the mutation sites, are synthesized. A set of primers,complementary to each other and spanning the mutation sites, are alsosynthesized. This set of primers (sense and anti-sense) contained theengineered mismatched nucleotides for site-directed mutagenesis. Twosets of PCR (30 cycles) were carried out using Pol-L/anti-sense andPol-R/sense primers, respectively, as PCR primers. The resulting DNAfragments were gel-purified. One tenth of each purified DNA fragmentswere mixed together and another PCR reaction (10 cycles) was performedin the absence of primers. Finally, Pol-L and Pol-R were added to thereaction and 20 more cycles of PCR were performed. After digested withXba I and purified by agarose gel electrophoresis, the resulting DNAfragment was used to replace the corresponding Xba I to Xba I fragment(nucleotides 250 to 1990) in pCMVHBV. The nucleotide sequences of allprimers used in the site-directed mutagenesis experiments were publishedpreviously. (Yeh et al., Hepatology (2000), 31:1318-1326).

[0111] The plasmids, pCMV-LLTQ and pCMV-YIDD, were constructed by onestep of mutagenesis experiment. The other two plasmids, pCMV-YVDD-LMAQand pCMV-YIDD-LMAQ, in which mutations were located in two separatepositions, were constructed by a two-steps mutagenesis experiment. Anintermediate plasmid, pCMV-LMAQ, was first generated for the second stepof experiment to generate the final plasmids. The nucleotide sequencesof all DNA plasmids were verified by DNA sequence analysis.

[0112] (2) Transfection of DNA Plasmids into HepG2 Cells

[0113] HepG2 cells were maintained in minimal essential medium (MEM)containing 10% fetal bovine serum. The standard CaPO₄ precipitationmethod was used to transfect cells. Cells were glycerol shocked for 1min at 5 h after transfection. The culture medium was changed on thesecond day and desirable concentrations of YKG were added. The cellswere harvested 24 hrs later.

[0114] (3) Isolation of HBVDNA and Southern Blot Analysis

[0115] HBV-DNA was analyzed by southern blot. Extraction of HBV DNA fromHepG2 cells was carried out according to a previously described protocolwith minor modification. (Yeh et al., J. Med. Virol. (1998), 55:42-50).Briefly, cells were trypsinized from the dish, rinsed withphosphate-buffered saline twice and lysed with 0.5 ml of TBS (10 mMTris-HCl [pH 7.2], 150 mM NaCl) containing 0.5% Nonidet P-40 (NP-40).After a brief centrifugation in a microcentrifuge at 1500×g, thesupernatant was transferred to a new tube and adjusted to 10 mmol/L ofMgCl₂. After digested with 100 μg/mL of DNase I for 30 min at 37° C.,ethylenediaminotetraacetic acid (EDTA) was added to a finalconcentration of 30 mmol/L to stop the reaction. To extract HBV DNA, anequal volume of buffer containing proteinase K (50 mM Tris-HCl [pH 7.2],1 mM EDTA, 1% SDS, 0.45% NP-40, and 200 μg/ml protease K) is added andthe mixture is incubated for 3 h at 55° C. After two extractions withequal volumes of phenol-chloroform and one extraction with chloroform,the DNA is precipitated with acid ethanol and dissolved in TE buffer (10mM Tris [pH8.0], 1 mM EDTA).

[0116] To detect HBV DNA, the extracted samples were loaded onto a 1%agarose gel for electrophoresis before blotted onto a nitrocellulosemembrane. HBV DNA is detected by hybridization with adigoxigenin-labeled probe. The method of labeling the probe as well asthe sensitivity, specificity and effective range for quantifying HBV DNAwith this probe are described elsewhere.

[0117] Result:

[0118] The effect of YGK on replication of HBV was assessed by detectingHBV-DNA replication intermediates by electrophoresis followed bysouthern blot analysis after 24 hours of incubation of HBV incubation inHepG2 cells. The relative amounts of relaxed circular plus linear formsof HBV-DNA were measured by densitometry and the amount of HBV-DNAderived from cells without drug treatment (0 mg/ml of drug) was assignedas 1 fold.

[0119] The results show that YGK, at a concentration of 20 mg/ml,inhibited viral replication in widetype HBV (suppressed to 0.61 fold),M552I HBV mutant (suppressed to 0.76 fold), L528M/M552V HBV mutant(suppressed to 0.45 fold), and A529T HBV mutant (suppressed to 0.61fold).

[0120] In sum, it appeared that YGK could effectively suppressreplication of most of the lamivudine-resistant HBV mutants.

EXAMPLE 6 Efficacy of YGK and Lamivudine Treatment on Patients withChronic Hepatitis B (HBV)

[0121] The clinical research was conducted in Liuhua Hospital inShenzhen, China.

[0122] Purpose:

[0123] The clinical research studied the effects of YGK and Lamivudinetreatment, either alone or in combination, on the clearance of serumHBV-DNA and negativity of HBeAg in chronic hepatitis B patients.

[0124] Method:

[0125] I. Test Articles

[0126] YGK injection solution (5 ml/ampoule, containing about 2g of YGKextract) and YGK capsules (about 220 mg/capsule, containing about 134 mgof YGK extract and about 86 mg of starch) were supplied by GongmingPharmaceutical Co., Ltd. of Heilongjiang Province, China.

[0127] Lamivudine 100 mg tablet was purchased from Glaxo Wellcome.

[0128] II. Cases Selection

[0129] (1) Inclusion Criteria

[0130] (a) Patients diagnose according to the National Hepatitis Bdiagnostic standard of 1990.

[0131] (b) Serum HBV-DNA continue to be positive 3 weeks prior totreatment.

[0132] (c) Serum HBeAg and HBsAg continuously positive and fluctuatingor continuous abnormal ALT.

[0133] (2) Exclusion Criteria

[0134] (a) Male or female aging under 18 or over 60, pregnant women orwomen in lactation.

[0135] (b) Patient with cardiovascular, pulmonary, renal or hemophthisisrelated diseases or mental disorder.

[0136] (c) Patient who did not meet the above criteria, did not complywith dosing regimen, or with missing data are excluded.

[0137] III. Grouping and Treatment

[0138] (1) Grouping Method

[0139] A total of 135 patients who met the criteria were included inthis study. Patients were randomized and divided into group A, B and C.Group A was the treatment group with 48 patients while group B with 42patients and group C with 45 patients were positive control groups.Patient gender, age, and medical history among groups were statisticallyanalyzed to be insignificant (p>0.05) and considered to be comparable.

[0140] (2) Treatment Method

[0141] Each treatment period lasted for 4 weeks. The duration oftreatment was 32 weeks (8 treatment periods).

[0142] (a) Group A: Patients received 10 ml YGK injection solution (2ampoules) diluted with 500 ml 5% glucose by I.V once daily during thefirst 2 treatment periods; 6 YGK oral capsules (220 mg) three timesdaily throughout the 8 treatment periods; and 1 lamivudine tablet (100mg) once daily during the first 4 treatment periods.

[0143] (b) Group B: Patients received 1 lamivudine tablet (100 mg) oncedaily for 8 treatment periods.

[0144] (c) Group C: Patients received 10 ml YGK injection solution (2ampoules) diluted with 500 ml 5% glucose through I.V. once daily duringthe first 4 treatment periods; and 6 YGK oral capsules three times dailythroughout the 8 treatment periods.

[0145] IV. Observations and Evaluations

[0146] (1) Observe Items

[0147] (a) Hepatitis B biological markers: Serum HBV-DNA, HBeAg,Anti-Hbe, HbsAg, Anti-HBs were analyzed before treatment, at week 4, 8,16 and 32 during treatment and at week 24 and 48 after treatmenttermination.

[0148] Serum HBV-DNA was measured by polymerase chain reaction (PCR)using 9600 DNA Amplification Machine (PE corp., USA) and reagents fromE-li-kang Biotech. Corp., Zhejiang, Wenzhou, China. Serum HBeAg,anti-HBe, HBsAg, and Anti-HBs were determined by the Imx method usingAutomatic Quick Micro Immune Analytic Instrument and reagents fromAbbott Laboratories, USA.

[0149] (b) Hepatic functions: ALT was monitored at week 4, 8, 16 and 32during treatment and at week 24 and 48 after treatment termination.

[0150] (c) Adverse effects: Patients were constantly monitored by themedical staff for adverse effect throughout the treatment periods.

[0151] (2) Statistical Analysis

[0152] Data obtained at weeks 4, 8, 16, and 32 during treatment and atweeks 24 and 48 after treatment termination were analyzed by Chi-square(X²) and t-test. The statistical results obtained were used to evaluatethe therapeutic effects.

[0153] Results:

[0154] 1. Observations During Treatment—Serum Biological Markers

[0155] (1) After one treatment period (four weeks of treatment)

[0156] The findings after 4 weeks of treatment are shown in Table 6.TABLE 6 Comparison of Curing Effects after 4 Weeks of Treatment SubjectALT Clearance of Seroconversion Seroconversion Seroconversion GroupNumber (normal) HBV-DNA of HBeAg of anti-Hbe of HBsAg Group A 48 47  46 9 9 2 Group B 42 28**  0**  0**  0** 0 Group C 45 38*  16** 3 3 0

[0157] From Table 5, it was obvious that there was significantimprovement in patient ALT level after 4 weeks of treatment. 97.9% ofGroup A patients (47/48) had normal ALT as compared to 66.6% of Group B(28/42, X²=15.75, p<0.01) and 84.4% of Group C (38/45, X²=5.39, p<0.05).Seroconverison of HBV DNA to negative in group A (95.8%, 46/48) wassignificantly higher than Group B (X²=82.35, p<0.01) and Group C(X²=38.04, p<0.01). Seroconversion of HBeAg to negative in group A(18.8%, 9/48) was significantly higher than group B (X²=8.55, p<0.01)but not significantly higher than Group C (X²=3.07, p>0.05).Seroconversion of HBsAg to negative in Group A was 4.2% (2/48) which washigher but not statistically significant than Groups B and C (X²=1.80and 1.95, respectively, p>0.05). None of the patients in the lamivudinepositive control group (Group B) had shown any effect on seroconversionof HBV DNA.

[0158] (2) After two treatment periods (eight weeks of treatment)

[0159] The findings after 8 weeks of treatment are shown in Table 7.TABLE 7 Comparison of Curing Effects after 8 Weeks of Treatment.Clearance Subject of HBV- Seroconversion Seroconversion SeroconversionSeroconversion Group Number DNA of HBeAg of anti-HBe of HBsAg ofAnti-HBs Group A 48 48  18 18 9  0 Group B 42  0**   0**   0** 0** 0Group C 45 16** 11 11 0** 0

[0160] As shown in Table 7, all patients of Group A had total clearanceof HBV DNA while Groups B and C remained unchanged. Seroconversion ofHBeAg to negative in Group A patients increased to 37.5% (18/48) whichwas significantly higher than Group B (X²=19.62, p<0.01) but notsignificantly higher than Group C (X²=1.86, p>0.05). Seroconversion ofHBsAg to negative in Group A patients increased to 18.7% (9/48) whilegroup B and C did not show any seroconversion, X²=8.55 and 9.39,respectively, with p<0.01. None of the patients in the lamivudinepositive control group (Group B) had shown any effect on seroconversionof HBV DNA.

[0161] (3) After four treatment periods (sixteen weeks of treatment)

[0162] Treatment of the YGK injection solution for Group A patients hadbeen discontinued after the 8^(th) week. The findings after 16 weeks oftreatment are shown in Table 8. TABLE 8 Comparison of Curing Effectsafter 16 Weeks of Treatment. Clearance Subject of HBV- SeroconversionSeroconversion Seroconversion Seroconversion Group Number DNA of HBeAgof anti-HBe of HBsAg of Anti-HBs Group A 48 48  23 23 18 5 Group B 4226**   2**   0**   0** 0 Group C 45 36** 14 14   0** 0

[0163] As shown in Table 8, it is apparent that the HBV DNA for patientsof Group A remained negative even with the discontinuation of the YGKinjection solution after the 8^(th) week. For Groups B and C, 61.9%(26/42) and 80% (36/45) of the patients, respectively, had shownnegative HBV DNA. However, these remained significantly (p<0.01) lowerthan that of Group A (X²=22.32 and 10.60, respectively, for Groups B andC). HBeAg seroconversion for Group A had increased to 47.9% (23/48)which was significantly higher than the 4.8% (2/42) of Group B(X²=20.79, p<0.01) but not significantly higher than the 31.1% (14/45)of Group C (X²=2.79, p>0.05). HBsAg seroconversion for Group A hadincreased to 31.1% (18/48) which was significantly higher than Groups Band C (X²=19.62 and 7.07, respectively, p<0.01). HBV DNA in Group Bpatients had shown relatively limited seroconversion when compared toGroup A.

[0164] (4) After eight treatment periods (thirty-two weeks of treatment)

[0165] Treatment of the lamivudine tablets for Group A patients andtreatment of the YGK injection solution for Group C patients had beendiscontinued after the 16^(th) week. TABLE 9 Comparison of CuringEffects after 32 Weeks of Treatment. Clearance Subject of HBV-Seroconversion Seroconversion Seroconversion Seroconversion Group NumberDNA of HBeAg of anti-HBe of HBsAg of Anti-HBs Group A 48 48 39 39 24 12 Group B 42  38*   4**   4**   1** 1 Group C 45 42 28* 28* 16 7

[0166] As shown in Table 9, it is apparent that at the end of week 32the patients in Group A continued to be negative for HBV DNA, eventhough they only took the YGK capsules since the end of week 16. HBV DNAnegative conversion for Group B has increased to 90.5% (38/42) which wasstill significantly lower than Group A (X²=4.77, p<0.05). Group C HBVDNA seroconversion had increased to 93.3% (42/45) which was comparableto Group A (X²=3.35, p>0.05). HBeAg seroconversion for Group A continuedto increase to 81.3% (39/48) which was significantly higher than Group B(9.5%, 4/42, X²=46.26, p<0.01) and Group C (62.2%, 28/45, X²=4.19,p<0.05 HBsAg seroconversion for Group A had increased to 50% (24/48)which was significantly higher than Group B (X²=25.38, p<0.01) but notsignificantly higher than Group C (X²=1.95, p>0.05). The results showedthat HBV DNA negative seroconversion for Groups A and C was far betterthan Group B.

[0167] 2. Adverse Effects

[0168] Group A patients had no noticeable side effects associated withthe treatment regime except the common soreness at the intravenousinjection and dry mouth.

[0169] After 16 weeks of treatment, 8 of the 42 patients in Group Bshowed adverse effects, such as anorexia, abdominal discomfort andfluctuating ALT levels. However, the symptoms disappeared after 2 moreweeks of treatment.

[0170] After 8 weeks of treatment, 17 of the 45 Group C patients showeddry mouth, abdominal discomfort and fluctuating ALT levels. However, thesymptoms disappeared after continuation of treatment and the liverfunction returned to normal.

[0171] 3. Treatment Follow-up TABLE 10 Serum biological markers at weeks24 and 48 after treatment termination. Clearance of SeroconversionSeroconversion Seroconversion Seroconversion Subject HBV-DNA of HBeAg ofanti-HBe of HBsAg of Anti-HBs Group Number 24W 48W 24W 48W 24W 48W 24W48W 24W 48W Group A 48 48 48 42  42  42  42  29 29 29  29  Group B 42 38*  26**  4**  0**  4**  0**   1**   0**  1**  0** Group C 45 42 4232* 32* 32* 32* 18 18 11* 11* [please verify]

[0172] As shown in Table 10, it is apparent that all Group A patientsusing the combination treatment of YGK injection solution, YGK capsulesand lamivudine remained negative of HBV DNA at week 24 and 48 aftertreatment termination. Three more patients had tested HBeAg negative inaddition to the 39 patients tested negative during treatment period,resulting in a 6.2% increase of the seroconversion rate to overall 87.5%(42/48). Five more patients had tested HBsAg negative in addition to the24 patients tested negative during the 32-week treatment, resulting in a10.4% increase of the seroconversion rate to overall 60.4% (29/48).

[0173] For Group C, no additional HBV DNA seroconversion was observedafter the termination of treatment. Four more patients showed HBeAgseroconversion and increased the seroconversion rate by 8.9% to overall71.1%, however this was still significantly lower than Group A (X²=3.91,p<0.05). Two more patients showed HBsAg seroconversion and increased theseroconversion rate by 4.4% to overall 40%, which was not significantlydifferent from Group A (X²=3.81, p>0.05).

[0174] For Group B, 12 of the 38 patients who were HBV DNA negativeduring the treatment periods had relapsed and showed positive for HBVDNA at week 48 after treatment termination. Overall seroconversion ratedecreased to 61.9% (26/42) which was 28.6% lower than that at the end ofthe 32-week treatment. Those few patients showing seroconversion ofHBeAG and HBsAg at the end of the 32-week treatment had all relapsed topositive at week 48 after termination of treatment.

[0175] Conclusion and Discussion:

[0176] The curing effects of the treatment regime on chronic hepatitis Bare evaluated by continued clearance of HBV-DNA in serum and livertissue, clearance of HBsAg and production of anti-HBs, suppression ofHBV replication, clearance of HBeAg and production of anti-HBe,significant improvement of liver tissue damage as shown by the normallevel of serum aminotransferase, histological examination of dead livercells and indication of alleviated inflammation.

[0177] The clearance of HBV and suppression of viral replication aredifficult to achieve because of the complicated viral structure andreplication cycle of HBV and the close relationship between thecondition of the patient's immune system and the existent state of thevirus in vivo. Thus, an effective treatment regime for chronic hepatitisB must both attack on every possible stage of the viral replication toblock the viral replication and improve the immune system of thepatients including breaking the immune tolerance of HBV and adjustingimmune balance of the body.

[0178] The combined treatment of YGK and lamivudine of the presentinvention is able to achieve the dual goals of suppressing viralreplication and improving immune system function of the patient.Lamivudine, as a representative of the second-generation nucleosideanalogue, is very effective on inhibition of the activity of reversetranscriptase during HBV replication. Six-month of lamivudine treatmentis effective to decrease HBV-DNA in serum, and the rate of patientsturning negative can reach as high as 90.4%. However, there areshortcomings for the lamivudine treatment, i.e., lamivudine only cutsoff a single point in the viral replication cycle so that the rate forpatients turning negative in HBeAg after lamivudine treatment is verylow (9.5%), and a long period of lamivudine treatment is needed. Infact, the present study confirms that the patients treated withlamivudine have a high rate of turning positive in HBeAg after thecompletion of the treatment.

[0179] The two types of YGK formulations, YGK intravenous injectionsolution and YGK capsules, are effective in treating patients with HBV.YGK intravenous injection solution has direct anti-HBV effect. Accordingto the present ongoing research, YGK intravenous injection solution canblock the take-up of nucleotides by the virus during viral replicationso that the virus will become feeble and die. YGK capsules, on the otherhand, are effective for the protection of liver cells and adjustment ofthe immune function. The combined use of both the intravenous injectionsolution and the capsules of YGK can effectively suppress thereproduction of HBV and stimulate the recovery of liver functions.

[0180] The combined treatment of lamivudine and YGK has the advantagesof providing multiple antiviral functions on multiple phases of HBVreplication. First, lamivudine inhibits the enzymatic activity ofreverse transcriptase during HBV replication. Second, YGK intravenousinjection solution blocks the uptake of nucleotides by the virus duringDNA replication. Finally, YGK capsules adjust the body immune functionso as to stimulate the recovery of the liver.

[0181] The results of the present study demonstrates that the combinedtreatment of YGK and lamivudine could clear the serum HBV-DNA within 8weeks. This is based on the facts that 100% of the patients showednegative for HBV-DNA after 8 weeks. The combined use of the twoformulation forms of YGK has further implication for developingtreatment regime for HBV.

[0182] While the invention has been described by way of examples and interms of the preferred embodiments, it is to be understood that theinvention is not limited to the disclosed embodiments. On the contrary,it is intended to cover various modifications as would be apparent tothose skilled in the art. Therefore, the scope of the appended claimsshould be accorded the broadest interpretation so as to encompass allsuch modifications.

We claim:
 1. A method for treating patients with liver diseases comprising: co-administering to said patients an effective amount of a pharmaceutical composition and lamivudine, wherein said pharmaceutical composition comprises aqueous extracts of an entire plant of Herba Hedyotidis diffusae, a rhizome of Rhizoma Bistortae, a rhizome of Rhizoma Polygoni Cuspidati, and a ripe fruit of Fructus Schisandrae.
 2. The method according to claim 1, said pharmaceutical composition further comprising aqueous extracts of a rhizome of Rhizoma Menispermi, a root of Radix Scutellariae, a bovine biliary powder, a tuber of Radix Curcumae, a ripe fruit of Fructus Crataegi, and a root of Radix Notoginseng.
 3. The method according to claim 2, further comprising aqueous extracts of a ripe fruit of Fructus Lycii, a root of Radix Ginseng Rubra, a root of Radix Scorphulariae, a root of Radix Angelicae sinensis, and a root of Radix Astragali.
 4. The method according to claim 3, wherein said pharmaceutical composition is administered to said patients orally.
 5. The method according to claim 4, wherein said orally administered pharmaceutical composition is in a dosage amount of about 0.5 to 5 g of said aqueous extracts of said pharmaceutical composition per day per person.
 6. The method according to claim 5, wherein said dosage amount of said orally administered pharmaceutical composition is about 1 to 3 g of said aqueous extracts of said pharmaceutical composition per day per person.
 7. The method according to claim 3, wherein said pharmaceutical composition is administered by intravenous injection.
 8. The method according to claim 7, wherein said pharmaceutical composition is in a dosage amount of about 1 to 10 g of said pharmaceutical composition per day per person.
 9. The method according to claim 8, wherein the dosage amount of said pharmaceutical composition is about 3-5 g of said pharmaceutical composition per day per person.
 10. The method according to claim 3, wherein said pharmaceutical composition is concurrently administered orally and intravenously.
 11. The method according to claim 1, wherein said lamivudine is administered orally.
 12. The method according to claim 1, wherein said lamivudine is orally administered in a dosage amount of 50-500 mg per day per person.
 13. The method according to claim 12, wherein the dosage amount of said lamivudine is about 100 to 200 mg per day per person.
 14. The method according to claim 1, wherein said liver diseases comprises at least one which is selected from the group consisting of hepatitis, cirrhosis, and liver cancer.
 15. The method according to claim 1, wherein said liver diseases are caused by hepatitis B viral (HBV) infection.
 16. A pharmaceutical combination comprising a first dosage unit of said pharmaceutical composition of claim 3 and a second dosage unit of lamivudine.
 17. The pharmaceutical combination according to claim 16, wherein said first dosage unit of said pharmaceutical composition is at least one which is selected from the group consisting of oral dosage form of said pharmaceutical composition and intravenous injection solution of said pharmaceutical composition.
 18. The pharmaceutical combination according to claim 17, wherein said oral dosage form of said pharmaceutical composition is at the amount of about 0.1 to 2 g per administration and at about 2-4 times a day.
 19. The pharmaceutical combination according to claim 17, wherein said intravenous injection solution of said pharmaceutical composition is at the amount of about 1 to 10 g per administration and at about once a day.
 20. The pharmaceutical combination according to claim 16, wherein said lamivudine is about 50 to 500 mg per administration and at about once a day.
 21. The pharmaceutical combination according to claim 16, wherein said pharmaceutical composition is used to treat patients with liver diseases.
 22. The pharmaceutical combination according to claim 16, wherein said liver diseases are at least one selected from the group consisting of hepatitis, cirrhosis, and liver cancer.
 23. The pharmaceutical combination according to claim 22, wherein said liver diseases are caused by hepatitis B viral (HBV) infection.
 24. A method for preventing a relapse of hepatitis in patients after withdrawal of lamivudine treatment comprising; administering a pharmaceutical composition to said patients after the withdrawal of lamivudine treatment; wherein said pharmaceutical composition comprises aqueous extracts of an entire plant of Herba Hedyotidis diffusae, a rhizome of Rhizoma Bistortae, a rhizome of Rhizoma Polygoni Cuspidati, and a ripe fruit of Fructus Schisandrae.
 25. The method according to claim 24, said pharmaceutical composition further comprising aqueous extracts of a rhizome of Rhizoma Menispermi, a root of Radix Scutellariae, a bovine biliary powder, a tuber of Radix Curcumae, a ripe fruit of Fructus Crataegi, and a root of Radix Notoginseng.
 26. The method according to claim 25, said pharmaceutical composition further comprising aqueous extracts of a ripe fruit of Fructus Lycii, a root of Radix Ginseng Rubra, a root of Radix Scorphulariae, a root of Radix Angelicae sinensis, and a root of Radix Astragali.
 27. The method according to claim 26, wherein said pharmaceutical composition is administered orally or intravenously, or is concurrently administered orally and intravenously.
 28. The method according to claim 24, wherein said hepatitis is caused by hepatitis B viral (HBV) infection.
 29. A method for supressing a development of lamivudine drug resistance in patients comprising: adminstering a pharmaceutical composition to said patients; wherein said pharmaceutical composition comprises aqueous extracts of an entire plant of Herba Hedyotidis diffusae, a rhizome of Rhizoma Bistortae, a rhizome of Rhizoma Polygoni Cuspidati, and a ripe fruit of Fructus Schisandrae.
 30. The method according to claim 29, said pharmaceutical composition further comprising aqueous extracts of a rhizome of Rhizoma Menispermi, a root of Radix Scutellariae, a bovine biliary powder, a tuber of Radix Curcumae, a ripe fruit of Fructus Crataegi, and a root of Radix Notoginseng.
 31. The method according to claim 30, said pharmaceutical composition further comprising aqueous extracts of a ripe fruit of Fructus Lycii, a root of Radix Ginseng Rubra, a root of Radix Scorphulariae, a root of Radix Angelicae sinensis, and a root of Radix Astragali.
 32. The method according to claim 29, wherein said pharmaceutical composition is administered orally or intravenously, or is concurrently administered orally and intravenously.
 33. The method according to claim 29, wherein said lamivudine drug resistance is caused by a development of lamivudine-resistant hepatitis B virus (HBV) strains in patients. 